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Objective: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro. Methods: The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons. Results: shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Conclusions: SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.
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Adenoviridae , Apoptose , Células Estreladas do Fígado , RNA Interferente Pequeno , Humanos , Adenoviridae/genética , Anexinas/análise , Apoptose/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Células Estreladas do Fígado/citologia , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Mensageiro , RNA Interferente Pequeno/farmacologiaRESUMO
Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.
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Adenoviridae , Células Estreladas do Fígado , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Proteínas de Transporte , Proliferação de Células , Cortactina , Filaminas/genética , Células Estreladas do Fígado/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Interferente Pequeno/genética , Ratos , Vinculina/genéticaRESUMO
Objective: To investigate the dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. Methods: Rat liver fibrosis model was established by intraperitoneal injection of CCl(4). Rat liver tissue histopathological changes were detected by HE and Masson-trichrome staining. Immunohistochemical staining, Western blot and real-time fluorescent quantitative PCR were used to detect SHP2 protein and mRNA expression in rat liver tissue. One-way analysis of variance was used for the comparison of means between multiple groups, and the LSD test was used for further inter-group comparison. Results: CCl(4)-induced rat liver fibrosis model was successfully constructed, and with the extension of modeling time, the degree of liver fibrosis in rats were aggravated gradually. Immunohistochemical staining results showed that SHP2 was mainly expressed in the cytoplasm of rat liver tissues. With the aggravation of liver fibrosis, the number of cells with positive expression of SHP2 was aggravated gradually (P < 0.05). Western blot and real-time fluorescent quantitative PCR results showed that the expressions of SHP2 protein and mRNA in rat fibrotic liver tissues at different times in week 2, 4, 6, and 8 were higher in modeling than control group (P < 0.05), and was aggravated gradually with the liver fibrosis aggravation (P < 0.05). Conclusion: The expression of SHP2 protein and mRNA in the liver tissue of rats with CCl(4)-induced liver fibrosis increased gradually with the degree of liver fibrosis, and the degree of increase was consistent with the degree of liver fibrosis.
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Tetracloreto de Carbono , Cirrose Hepática , Animais , Tetracloreto de Carbono/toxicidade , Cirrose Hepática/induzido quimicamente , Proteínas Tirosina Fosfatases , Ratos , Ratos Sprague-DawleyRESUMO
It is more than 20 years since the first endovascular repair of thoracoabdominal aortic aneurysm was performed. Significantly reducing risk of morbidity and mortality compared with open aortic repair, the advent of endovascular repair has revolutionized the treatment of complex aortic disease. It is now the first-line treatment for most thoracoabdominal aortic aneurysm patients. However, spinal cord ischemia (SCI) remains one of the most threatening complications after the procedure, and significantly reduces overall survival. The pathophysiology of spinal cord ischemia remains unclear but may well be multifactorial. Risk factors for SCI include excessive aortic coverage, detriment to the collateral network through occlusion of the left subclavian or internal iliac arteries, perioperative hypotension and chronic renal failure. SCI could be predicted, prevented and ameliorated through the application of motor evoked potential, permissive perioperative hypertension, cerebrospinal fluid drainage (CSFD), preoperative or concomitant left subclavian artery (LSA) revascularization and some drugs. However, there are certain complications and contraindications for CSFD and arterial revascularization. As a result, we must balance the pros and cons of these invasive measures. So we summarize our clinical experience and propose the employment of LSA revascularization and CSFD in certain kinds of high-risk patients respectively. With the development of technology and preventive measures, we believe that SCI could be minimized in the forseeable future.
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Aneurisma da Aorta Torácica , Implante de Prótese Vascular , Procedimentos Endovasculares , Isquemia do Cordão Espinal , Aneurisma da Aorta Torácica/cirurgia , Implante de Prótese Vascular/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Humanos , Estudos Retrospectivos , Fatores de Risco , Medula Espinal/irrigação sanguínea , Isquemia do Cordão Espinal/etiologia , Isquemia do Cordão Espinal/prevenção & controle , Resultado do TratamentoRESUMO
STUDY DESIGN: Retrospective cohort study. OBJECTIVES: To analyze magnetic resonance imaging (MRI) evaluator agreement in dogs with spinal cord injury (SCI) caused by intervertebral disk herniation (IVDH) using semiautomated and manual lesion segmentation and to analyze the associations between MRI and functional outcome. SETTING: United States of America. METHODS: T2-weighted MRIs from dogs with SCI resulting from thoracolumbar IVDH were identified from a database. Evaluators categorized MRIs on the basis of the presence or absence of a T2-hyperintense spinal cord lesion in axial and sagittal images. A semiautomated segmentation algorithm was developed and used to estimate the lesion volume. Agreement between evaluators and between semiautomated and manual segmentation was analyzed. The relationships of qualitative and quantitative MRIs with behavioral functional outcome were analyzed. RESULTS: Axial images more commonly depicted lesions compared with sagittal images. Lesions in axial images had more consistent associations with functional outcome compared with sagittal images. There was imperfect qualitative agreement, and lesion volume estimation was imprecise. However, there was improved precision using semiautomated segmentation compared with manual segmentation. CONCLUSION: Lesion volume estimation in dogs with naturally occurring SCI caused by IVDH is challenging, and axial images have important advantages compared with sagittal images. The semiautomated segmentation algorithm described herein shows promise but may require further refinement.
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Imageamento por Ressonância Magnética/métodos , Traumatismos da Medula Espinal/patologia , Algoritmos , Animais , Modelos Animais de Doenças , Cães , Feminino , Processamento de Imagem Assistida por Computador , Deslocamento do Disco Intervertebral/complicações , Deslocamento do Disco Intervertebral/patologia , Masculino , Reconhecimento Automatizado de Padrão , Estudos Retrospectivos , Traumatismos da Medula Espinal/etiologia , Vértebras TorácicasRESUMO
An E-band (60-90 GHz) multi-channel Doppler backscattering (DBS) system with X-mode polarization has been installed on the Experimental Advanced Superconducting Tokamak (EAST), which can measure the turbulence at five different radial locations simultaneously. This system can launch 31 fixed microwave frequencies in the range of 60-90 GHz with a 1 GHz interval into the plasma, and five probing signals are selected by employing a reference signal and multiple filters. During experiments, the frequency of the reference signal is tunable in the E-band, and the selected probing signals can be changed as needed without any other adjustments, which can be performed in one shot or between shots. Furthermore, the incident angle can be adjusted from -10° to 20°, and the wavenumber range is 4-25 cm-1 with a wavenumber resolution of Δk/k ≤ 0.35. Ray tracing simulations are employed to calculate the scattering locations and the perpendicular wavenumber. In this article, the hardware design, ray tracing, and initial results obtained from the EAST plasma will be presented.
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The cross-polarization scattering (CPS) system for magnetic fluctuation measurements in the Experimental Advanced Superconducting Tokamak (EAST) has been designed and installed. Different from the Doppler reflectometer (DR) system, the CPS system detects the perpendicular polarization of the electromagnetic wave induced by magnetic fluctuations BÌ. The CPS system in the EAST has been developed from the existing Doppler reflectometer system, and they are integrated together for simultaneous measurement of magnetic and density fluctuations. Ray-tracing simulations are used to calculate the scattering locations and the wavenumber coverage of the magnetic fluctuation for CPS. In the experiments, the CPS and DR system data were different in Doppler shift, amplitude, and spectrum broadening. In this article, the hardware design, the ray tracing, and the preliminary results of the system in the EAST are presented.
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A 5-channel Doppler backscattering system has been designed and installed in the Experimental Advanced Superconducting Tokamak (EAST). Through an I/Q-type double sideband modulator and a frequency multiplier, an array of finely spaced (Δf = 400 MHz) frequencies that span 1.6 GHz has been created. The center of the array bandwidth is tunable within the range of 75-97.8 GHz, which covers most of the W band (75-110 GHz). The incident angle can be adjusted from -4° to 12°, and the wavenumber range is 4-15 cm-1 with a wavenumber resolution of Δk/k ≤ 0.35. Ray tracing is used to calculate the scattering location and the scattering wavenumber. This article details the hardware design, the ray tracing, and the preliminary experimental results from EAST plasmas.
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Duchenne muscular dystrophy (DMD) is a fatal Xlinked muscle disorder caused by mutations in the dystrophin gene with a consequence of progressive degeneration of skeletal and cardiac muscle. Golden retriever muscular dystrophy (GRMD) is a spontaneous X-linked canine model of DMD with similar effects. Due to high soft-tissue contrast images, MRI is preferred as a non-invasive method to extract information corresponding to biological characteristics. We propose and evaluate non-invasive MRI-based imaging biomarkers to assess the severity of golden retriever muscular dystrophy (GRMD) using 3T and 4.7T MRI data of nine animals. These imaging biomarkers use first order statistics and texture (assessed by wavelets) in quantitative MRI (qMRI). In a leave-one-sampleout cross-validation framework, we use SVM to differentiate between young and old GRMD animals. The preliminary results show good differentiation between young and old animals for different qMRI sequences and based on a different selection of features.
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Distrofia Muscular Animal/diagnóstico por imagem , Distrofia Muscular de Duchenne/diagnóstico por imagem , Animais , Biomarcadores , Modelos Animais de Doenças , Doenças do Cão/diagnóstico por imagem , Cães , Imageamento por Ressonância Magnética , Músculo Esquelético/diagnóstico por imagemRESUMO
Based on a new technique, a tunable, multi-channel system that covers the Q-band (33-55 GHz) is presented in this article. It has a potential use of the Doppler backscattering system diagnostic that can measure the turbulence radial correlation and the perpendicular velocity of turbulence by changing the incident angle. The system consists primarily of a double-sideband (DSB) modulation and a multiplier, which creates four probing frequencies. The probing frequency enables the simultaneous analysis of the density fluctuations and flows at four distinct radial regions in tokamak plasma. The amplitude of the probing frequency can be adjusted by the initial phase of the intermediate frequency (IF) input from the double-sideband, and the typical flatness is less than 10 dB. The system was tested in the lab with a rotating grating, and the results show that the system can operate in the frequency range of 33-55 GHz with a Q-band multitude and that the power of each channel can be adjusted by the phase of the IF input of DSB.
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Duchenne Muscular Dystrophy (DMD) is a genetic disorder caused by dystrophin protein deficiency. Muscle biopsy is the gold standard to determine the disease severity and progression. MRI has shown potential for monitoring disease progression or assessing the treatment effectiveness. In this study, multiple quantitative MRI parameters were used to classify the tissue components in a canine model of DMD disease using histoimmunochemistry analysis as a "ground truth". Results show that multiple MRI parameters may be used to reliably classify the muscular tissue and generate a high-resolution tissue type maps, which can be used as potential non-invasive imaging biomarkers for the DMD.
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Distrofia Muscular de Duchenne , Animais , Biomarcadores , Progressão da Doença , Cães , Distrofina , Imageamento por Ressonância Magnética , Músculo EsqueléticoRESUMO
Doppler backscattering system can measure the perpendicular velocity and fluctuation amplitude of the density turbulence with intermediate wavenumber. An eight-channel Doppler backscattering system has been installed in the Experimental Advanced Superconducting Tokamak (EAST), which can probe eight different radial locations simultaneously by launching eight fixed frequencies (55, 57.5, 60, 62.5, 67.5, 70, 72.5, 75 GHz) into plasma. The quasi-optical system consists of circular corrugated waveguide transmission, a fixed parabolic mirror, and a rotatable parabolic mirror which are integrated with quasi-optics front-end of the profile reflectometer inside the vacuum vessel. The incidence angle can be chosen from 5° to 12°, and the wavenumber range is 2-15/cm with the wavenumber resolution Δk/k≤0.21. Ray tracing simulations are used to calculate the scattering locations and the perpendicular wavenumber. The dynamic range of this new eight-channel Doppler backscattering system can be as large as 40 dB in the EAST. In this article, the hardware design, the ray tracing, and the preliminary experimental results in the EAST will be presented.
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Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder. Therapeutic assessments currently require muscle biopsy to ascertain information about the status of disease progression. MRI shows potential to be used in place of muscle biopsy for therapeutic assessments. In this work, localized histological data and various MRI parameters were correlated in a canine model of DMD. The results indicate several MRI parameters may be useful as biomarkers of disease progression.